Fructose obtained by enzymatically isomerizing dextrose to fructose is extensively used by the food industry as a sucrose replacement.
Substantial fructose production costs are encountered by the need to frequently replace spent or deactivated glucose isomerases. Enhanced fructose productivity by glucose isomerase is a desirable goal. Extensive research efforts have been expended towards obtaining maximum fructose productivity with the lowest possible amount of glucose isomerase. Many researchers have deemed the solution to the problem as being simply a matter of discovering a stable glucose isomerase. In this endeavor, the art has screened, mutated and prepared a multitude of different glucose isomerase preparations. In testing the efficacy and susceptibility of these glucose isomerases to deactivation, the art has come to the realization that isomerases derived from different microbe sources possess different enzymatic characteristics. Optimum isomerization conditions such as pH and temperature, isomerase activators (e.g., metal ion activators such as Co.sup.++, manganese, etc.) and other processing variables will depend upon particular glucose isomerase type. In general, a greater disparity in isomerization conditions occurs between isomerases derived from a different genera.
Immobilized isomerases are more stable than isomerases in a water-soluble or unbound form. In general, immobilized glucose isomerases are better suited for commercial operations since they may be continuously used in batch or continuous operations until exhausted. Upon exhaustion, these isomerases are replaced with fresh immobilized isomerase. Most isomerization reactions are conducted at temperatures and pHs which optimize the rate at which the isomerase converts dextrose to fructose. Similar to other enzymes, isomerases are usually most stable against inactivation (including heat inactivation) and possess a higher enzymatic activity when used at their optimum isomerization pH. Glucose isomerases presently used in the commercial production of fructose containing syrups characteristically exhibit improved stability and activity when the isomerization process is conducted in the presence of Co.sup.++ ions. Cobaltous salts are occasionally added to feed syrups for this purpose. It would be desirable to achieve a higher productivity without necessitating cobaltous ions.
A suggested glucose isomerization processing modification is to increase the isomerization reaction temperature as isomerase activity decreases. An increase in the reaction temperature will accelerate the rate of fructose production as well as the rate at which the isomerase deactivates. The net effect is to lower the total fructose yield produced by the isomerase.
Isomerases reportedly produced by organisms belonging to the Bacillus genera include Bacillus stearothermophilus ATCC 31265, NRRL B-3680, NRRL B-3681 and NRRL B-3682; Bacillus sp. NRRL B-5350 and NRRL B-5351; Bacillus megaterium ATCC 15450; Bacillus fructosus ATCC 15451, 35c. (e.g., see U.S. Pat. Nos. 3,826,714 by Suekane et al. and 3,306,752 by K. Ueda, West German printed patent application No. 2,164,342 filed under Ser. No. P 2,164.342.5 on Dec. 23, 1971 by K. Aunstrup et al., Agri. Biol. Chem., Vol. 31, No. 3, pages 284-292, 1967 by Danno et al.). Heat or chemical treatment of viable cells containing intracellular isomerase, encapsulation, complexing with natural and synthetic polymers, immobilization within a binder matrix and numerous other means for immobilizing isomerases have been suggested. Exemplary methods for immobilizing isomerases and enhancing enzymatic stability are disclosed in J. Appl. Chem. Biotechnol. 1974, 24, 663-676 by Kent et al. Agri. Biol. Chem., Vol. 30, No. 10, pages 1015-1023, 1966 by S. Yoshimura et al. (e.g., see Holland patent application No. 73/12525 filed Sept. 11, 1973 and assigned to Novo Terapeutisk Laboratorium, British Patent Specification No. 1,274,158, U.S. Pat. Nos. 3,821,082 by Lamm et al., 3,779,869 by Zienty 3,694,314 by Lloyd et al., 3,788,945 by Thompson et al., and British Patent Specification No. 1,356,283 by Monsanto Co., etc.).
A recent article entitled "Sweetzyme--A New Immobilized Glucose Isomerase," die Starke 27, Jahrg 1975/Nr. 7, pages 236-241 discloses immobilized isomerases of a Bacillus coagulans origin. This article defines glucose isomerase productivity as the combined effect of activity and stability. At the more neutral pHs, cobalt is deemed essential for fructose productivity. In order to achieve optimum fructose syrup productivity in the absence of cobalt ions, the authors conclude that it is necessary to conduct the continuous isomerization reaction at a relatively high alkaline pH. For a continuous operation (e.g., column isomerization) involving a short contact and reaction time between isomerase and syrup, optimum productivity (in the absence of Co.sup.++) is reportedly achieved at a pH above 8.0 (e.g., 8.1-8.5), a 40-45% solids level and 65.degree. C.